The goal of this project is to understand the steps in development and maturation of the cholinergic neuronal phenotype. Toward this goal, the first aim is to generate a panel of monoclonal antibodies (MAbs) which specifically identify cholinergic neurons. Two different approaches, fluorescence activated cell sorting of identified neurons following backfilling from targets, and a novel immunosuppression method, will be used to optimize obtaining such antibodies. A related aim is to find MAbs which distinguish subclasses of cholinergic neurons, and correlate these marked populations with lineage, target and trophic requirements. The second specific aim is to determine the time course and order of acquisition of epitopes common to all cholinergic neurons, and correlate them to the time of appearance of choline acetyltransferase (CAT), the acetylcholine synthesizing enzyme. If different populations of cholinergic neurons all undergo a similar basic plan of development, this may be reflected in the sequential appearance of cholinergic markers. Thus the antigenic profile of a neuron may be an accurate means of staging its state of differentiation. Markers which are expressed before the appearance of CAT may recognize, and be used to purify, cholinergic precursor cells for in vitro studies of development. The third aim is to identify potentially dual-functional populations of cholinergic neurons. Double-label immunofluorescence studies will allow direct identification of neurons with both CAT and neuropeptide immunoreactivity. It will then be important to determine the order of appearance of the two "transmitters" during development, and study the regulation of each transmitter type. Currently, the presence of CAT is the best means of identifying a cholinergic neuron; any new markers specific for this phenotype will be very useful for studying this class of neurons. Cell surface markers will be particularly useful for selecting and sorting out pure populations of cholinergic neurons for in vitro studies. The long term aim of this research is to study the interactions of cholinergic neurons with environmental and target derived factors in controlled conditions of tissue culture, and in vivo.